Thousands of recreational beaches along United States coasts have experienced water quality advisories or closures due to elevated levels of fecal indicator bacteria, such as Escherichia coli (E. coli). However, this methodology may not reflect the number of actual pathogens present or the potential human health risk. Also, little is known about the survival of biological contaminants in the beach sand, which may impact monitoring results. In order to validate use of E. coli as an indicator organism, it is important to understand the physiological mechanisms for adaptation and survival. This proposal encompasses three major aims. Aim 1 focuses on continuation of a 4-year study to evaluate the E. coli burden at a heavily trafficked urban beach on Lake Michigan. Bacterial counts will be determined throughout the year. Clonal typing will be performed to determine the extent to which selected populations from contamination sources can adapt to and colonize the beach sand habitat. Aim 2 investigates the induction and modulation of two physiological responses that may confer protection against desiccation stress in sand; namely trehalose synthesis and shifts in porin protein expression. Sand model plots will be loaded with a beach sand E. coli isolate and placed on an environmental green roof for exposure to the same conditions as nearby beachfronts. Survival outcomes for E. coli will be determined under fluctuating desiccation/rehydration conditions and in the presence or absence of resident microbiota. The gene products for porin protein ompC and the end-product of the ots operon (trehalose) will be measured in sand eluant samples taken throughout the study. Aim 3 focuses on long-term survivorship of selected E. coli from the beach site and for isolates loaded in short term (<7 days) and long-term (>14 days) sand plot models. Expression of fitness advantage traits (e.g. osmotic tolerance) will be evaluated under environmental and controlled growth conditions of varying temperature and desiccation and in competition assays with known laboratory strains. Throughout all aims we will examine modulation of genes associated with growth phase transitions (e.g. rpoS, dps, katE). Findings from these studies will provide public health officials with more contextual information in which to interpret water monitoring data, and will allow beach managers to formulate new strategies for abatement of coliform pollution in recreational waterfronts. [unreadable] [unreadable] The cellular mechanisms involved in the survival of the fecal indicator organism E. coli in beach sand, a habitat of increasing interest, are relatively unknown. Almost all designated public beaches in coastal regions will have monitoring programs for fecal pollution implemented by 2007. Understanding the environmental reservoirs and determinants of E. coli die-off will be important for interpreting monitoring results of beaches and discerning whether elevated E. coli levels reflect human health risk. [unreadable] [unreadable] [unreadable] [unreadable]